1. Field of the Invention
This invention relates to the detection and quantification of small quantities of man-made chemicals such as herbicides, insecticides and fertilizers in soil, water and plant samples. This invention also relates to the design of antigens capable of eliciting immune responses which in turn are capable of producing antibodies specific to particular substances.
2. Description of the Prior Art
Trifluralin, a member of the dinitro analin family, is a well known and widely used herbicide. It is particularly effective against Kochia and pigweeds. Unfortunately, residues of trifluralin can persist in soils for many months after its use as a herbicide. These residues can kill or seriously injure subsequently planted crops. Winter wheat is particularly susceptible. Hence, farmers are particularly concerned with determining the presence and concentration of trifluralin residues in their soils, particularly in the period just prior to planting.
The most commonly used techniques for making these determinations have a number of drawbacks. For example, those bioassay procedures which can be employed right on the farm can only detect those upper range of trifluralin levels which are known to injure specific crops such as for example winter wheat. Moreover, the farmer needs a great deal of skill, time and discipline to carry out these bioassays in a manner which is likely to produce reliable results. Oat seeds must be planted in pots and maintained under carefully controlled conditions for at least twenty days. After seed germination, each pot is thinned to about three plants of uniform size. These plants are then uprooted and washed clean of any soil and weighted to a high degree of accuracy. The resulting weights are analyzed statistically to provide a linear representation of trifluralin residue in the samples.
Aside from the weighing and statistical skills required for accurate results, these bioassays are hampered by the fact that their limit of sensitivity is about 0.10 lbs/acre. Unfortunately, trifluralin concentrations lower than 0.10 lbs/acre are known to be harmful to certain crops, particularly winter wheat. Hence bioassays, if done accurately, can only warn the farmer that trifluralin concentrations are too high. They can not however, tell whether the concentrations are far enough below 0.10 lbs/acre to assure a safe planting.
Alternatively, farmers can send soil samples to commercial laboratories. These commercial laboratories may carry out bioassays in a more rigorous fashion than the farmer or they may use more sophisticated techniques such as gas chromatography or high pressure liquid chromatograph. However, this course of action is both expensive and time consuming since many of the laboratory techniques are complex. For example, the U.S. Environmental Protection Agency recently proposed a complex series of methods for analyzing pesticide pollutants. These procedures include many industry and contractor-developed analyses, and several analyses developed by the EPA's own Environmental Monitoring Support Laboratory. One of these methods uses gas chromatography for purification of the sample. The residue is detected by using an electron capture (.sup.63 Ni) detector. Detection limits for trifluralin range from 10.sup.-13 to 10.sup.-12 g. However, accuracy and precision studies suggest that samples normally contain these compounds in parts per billion, i.e., levels 10.sup.-3 to 10.sup.-5 times higher than the detection limit. In this range, high pressure liquid chromatogrphy coupled with ultra-violet detection is an alternative to the gas chromatography analysis which has, in the past, been the accepted standard for trifluralin analysis. In any case, accuracy and expense are important factors that should be considered when these techniques are used since both require a great deal of purification before the sample can be quantitated. Organic material must be removed and, in some cases, several solvent systems with a complex reflux apparatus are needed. However, the overall results realizable using these techniques can provide a very accurate determination (sensitive to the low parts per billion) of the residual herbicide in the soil sample. Again, however, the cost of the equipment, materials, and labor needed to carry out these tests are considerably higher than with bioassays. Samples sent to a laboratory will currently cost between $50.00 and $100.00 each, depending on which methods are used; and it could take several weeks or more to get back the results. Moreover, several samples per field must be assayed to determine overall residual herbicide levels and the cost, therefore, often becomes prohibitive. Therefore, less costly and more rapid alternatives for testing for trifluralin residues would be a welcome addition to these known test techniques.
Until recently, immunological techniques have been largely confined to medical and veterinary research, however, lately there have been some noteworthy successes in the field of horticulture, especially in the area of identification and control of various plant viruses. By way of background, the so-called "microplate enzyme linked immunosorbant assay" (ELISA) has had a significant impact on both the identification and control of several harmful plant pathogens. It is inexpensive, reliable and quite sensitive. Prior to the advent of this technique, while certain immunological methods were tried from time to time but, they proved to be of little value in agriculture, perhaps because of the cost and the high degree of technical expertise needed to carry them out. This is no longer true and the field of immunology holds great promise for agriculture. Unfortunately, realizing that immunological techniques are applicable does not solve the fundamental problem of designing and creating antigens which will evoke a desired immune response and result in the production of an antibody highly specific to any given chemical which is to be detected.
By way of further background in another area of immunology which relates to this invention, haptens are molecules that by themselves are too small, or for some other reason will not elicit an immune response. These smaller or non-immunogenic molecules must, therefore, be linked to a large protein before the resulting substance can elicit an immune response. Generally speaking, molecules with a molecular weight less than about 1,000 daltons need to be attached to a carrier protein in order to evoke such a reponse. Proteins with substances linked to their side-chains are referred to as "conjugated proteins". The side groups and the protein together make up the conjugated compound that will determine the antigenic response. It is essential that a functionality be present on the molecule of interest (the hapten) which will react with a protein. In addition, of course, it must assume a specifically to the targeted compound without responding to the presence of other analogous compounds. Despite the obvious difficulties associated with attempting to hypothesize the chemical make-up of an antigen that would produce a specific antibody, to say nothing of producing one having such a structure, we have succeeded in synthesizing an antibody which is highly specific to trifluralin.
U.S. Pat. No. 4,530,786 (the 786 patent), issued to the Applicants herein, is believed to be the closest prior art to the teachings of this patent application and the teachings of the 786 patent are specifically incorporated into this patent disclosure. The 786 patent teaches detection and quantification of small amounts of atrazine, i.e., 2-chloro-4-ethylamino-6-isopropylamino S-triazine: ##STR1## The detection and quantification of atrazine disclosed in the 786 patent was accomplished by first substituting a soluble, straight chain amino acid having at least four carbon atoms at the 4 or 6 position of the atrazine molecule so as to leave the chlorine exposed at the 2 position along with one of the remaining amino groups located at either the 4 or 6 position. The resulting substitution product acts as a hapten which is then conjugated at the site of the substituted amino acid group with a lysine-rich protein. This conjugated produce was used as an antigen to elicit an immune response which produces an antibody which is highly specific to atrazine.
However, as is well known in the art, and as is noted in the 786 patent, "realizing that immunological techniques are applicable does not solve the fundamental problem of designing and creating the antigen which will evoke the desired immune response and result in the production of an antibody specific to the molecule to be assayed". Theses words notwithstanding, Applicants, in attempting to design other antigens based upon some of the premises assumed in designing the antigen disclosed in the 786 patent, have discovered another antigen and other design premises which do not follow from those applicable to the atrazine specific antibody disclosed in the 786 patent, but which nonetheless have produced another very different, and distinctly useful, antibody. From the antigen design perspective, we postulated in the research which led to the 786 patent that the haptenic substitution product, like the atrazine, needed to have a chlorine atom exposed at the 2 position, and that the amino substituted atrazine molecule should be conjugated at either the amino ehtyl group in the 4 position or at the amino isopropyl group in the 6 position. We also postulated that the remaining amino group had to be left exposed for detection by the response mechanism. Moreover, the amino acid used in the 786 patent disclosure was postulated to require at least four carbon atoms.
These problems did not however remain valid with respect to the design of an entirely different antigen which we have found to be capable of eliciting the production of an antibody which is highly specific to trifluralin--a member of the dinitro analine family and a widely used herbicide. Trifluralin has the structural formula: ##STR2##
The structure of trifluralin as well as its known chemical reactivities are such that the chemical and biological reasoning used in designing the antigen disclosed in the 786 patent were not applicable. For example, trifluralin, unlike atrazine, has no chlorine in its 2 position; in fact trifluralin has no chlorine anywhere in its structure. Similarly, upon substitution of a soluble, straight chain amino acid to trifluralin, the resulting molecule, unlike atrazine, has no unreacted, exposed amino group anywhere in its structure. Nonetheless, the antigen disclosed herein is capable of evoking the production of another distinct antibody which is highly specific to trifluralin. Moreover, the trifluralin can undergo a reaction with a soluble, straight chain amino acid having as few as one carbon, as opposed to the four carbon atom lower limited established in the case of the atrazine substitution disclosed in the 786 patent.
Those skilled in the art will of course appreciate that once an antibody such as the one disclosed in this patent application has been produced, it can form the basis for a number of different assay procedures. For example, it could be used in microplate enzyme linked immunosorbant assay (ELISA) procedures. However, for reasons having to do with relative costs and the level of technical skills required, radioimmunoassay procedures are highly preferred for the practice of this invention. They are especially suited for detecting and quantifying the presence of trifluralin in such biologically important materials as plants, soil and water. Therefore, a little background information about radioimmunoassays also will serve to place our invention in further context with the prior art.
The basic principle of a radioimmunoassay is a competitive reaction of an antigen, such as the antigen disclosed in this patent application, as well as a radiolabeled form of that same antigen, for binding sites on an antibody which is highly monospecific to the antigen. Therefore a successful radioimmunoassay requires: (1) a specific, sensitive, high affinity antibody against the antigen that is to be measured; (2) a radioactivity labeled antigen of high specific activity that will react with the antibody with a comparable affinity to that of the unlabeled antigen; (3) availability of a suitable preparation of the antigen for use as a standard in the assay such that it will react with the antibody in a manner identical to that of the antigen in the sample being assayed; and (4) a simple, reproducible method of separating the antigen bound to the antibody from the free labeled antigen. A number of variations in the procedures used to carry out such assays are known to the art, see for example Freeman, Samuel and Gold, Phil, Clinical Immunology, 2nd Ed, 590-599, 1976. In all cases however, in order for a given radioimmunoassay test to be of use, it is essential that the given antigen/antibody dissociation reaction be specific for the anitgen to be measured.
However, nonspecific antibody production is often the rule rather than the exception. It often occurs in biological situations where structures of more than one substance are similar and/or where overlap, even in biological activities, may occur to some greater or lesser degree. By way of further background example, one such overlap situation is seen in the assay systems used to detect the glycoprotein pituitary hormones TSH, LH and FSH. Each of these hormones contains an identical alpha subunit. Biological activity, however, resides in the beta chains, which are slightly different in each of the three hormones. Hence, significant cross activity of these substances may occur with any given antibody. Moreover, injections of many substances during immunization procedures can result in formation of multiple antibodies that theoretically, and in fact, combine with any of several immunologically active sites on the antigen molecule. Other problems also exist. For example, it is also well known that antibody molecules of different specificities are often produced against any given hapten-protein complex. Consequently, finding antibodies with highly specific recognition capabilities, such as the one disclosed in this patent application, remains largely a difficult, empirical tasks.
Those skilled in the art will also appreciate that there are primarily two ways in which production of antibodies with sufficient affinity for use in radioimmunoassays have been achieved. For proteins and polypeptides of a molecular weight greater than about 5,000 daltons, the inherent immunogenicity of the substance itself usually results in production of sufficient antibody for development of an assay system. However, for biologically active compounds of lower molecular weights (haptens), especially those with molecular weights less than about 1000 daltons, such as the one disclosed in this invention, antibody production can usually only be achieved by conjugation of the low molecular weight haptenic material with a protein having significantly greater molecular weights. In other words, conjugation is essential for eliciting an immune responses to small substances that have no inherent immunogenicity and therefore cannot induce antibody formation. It is well known in the art that a variety of proteins can be used for conjugation of low-molecular-weight haptens such as the trifluralin based antigen disclosed in this patent application. Some of the more common protein materials used for this purpose include bovinve serum albumin, ovalbumin, thyroglobulin and fibrinogen. Here again however, such variations in these known conjugation procedures and materials do not in any way negate the need for antibody specificity in whatever assay procedure is ultimately employed.